We have developed a new cell culture method (Feeding Culture) for colony formation human leukemic marrow cells (L-CFU), which can be used as a quantitative assay for leukemic stem cells. With the previous grant from USPHS, we have solidified the value of this culture assay by a number of quantitative (e.g., successful colony growth, over 73% in 80 patients with acute nonlymphocytic leukemia), and qualitative studies (e.g., chromosome analysis). Our preliminary study in a limited number of patients on the in vitro chemotherapy effect on L-CFU in comparison with that on CFU-C (normal granulocytic progenitor cells) showed an extremely good correlation with clinical chemotherapy response (p less than 0.001). We plan to extend this study to a larger number of patients with a variety of in vitro chemotherapy exposures (change in schedules and drug combinations, and trials of new drugs, etc.) in order to find the best in vitro predictive system for clinical chemotherapy response and also to find new improved chemotherapy regimens. Cell-sorting by Flow microfluorometry with the use of supravital DNA stain (H33342) followed by Feeding Culture for L-CFU with or without in vitro chemotherapy exposure will be done in order to improve understanding of leukemic stem cell kinetics and to facilitate the development of the new improved chemotherapy regimens. We will characterize and quantitate the effects of in vitro growth regulating factors (L-ascorbic acid, colony stimulating factor and leukemic suppressing factor) found to be important for L-CFU. This will enable better utilization of our culture assay and may also lead to new mode of controlling leukemia.